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Keywords:

  • Drosophila;
  • glia;
  • eye disc;
  • development

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORMATION OF THE DROSOPHILA EYE
  5. GLIAL CELLS IN THE EYE
  6. MIGRATION OF GLIAL CELLS ONTO THE EYE DISC
  7. NEURON–GLIA INTERACTION IN THE EYE DISC
  8. REFERENCES

The Drosophila compound eye comprises about 750 individual ommatidia arranged into an almost crystalline array. The eye is not needed for viability and thus served as a favorite model organ to decipher many signaling systems controlling diverse aspects such as cell fate allocation or cell-cycle control. Here, we review that the Drosophila eye can also serve to study the interaction between neurons and glial cells. In the Drosophila eye, all glial cells originate from the brain lobes and need to migrate onto the larval eye disc as neurogenesis is initiated during the third instar stage. Although we do have a relatively good understanding of the sequential progression of neurogenesis in the eye disc, we are still at the beginning in our dissection of the molecular pathways orchestrating the coordinated development of neurons and glial cells. © 2011 Wiley Periodicals, Inc. Develop Neurobiol 71: 1310-1316, 2011


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORMATION OF THE DROSOPHILA EYE
  5. GLIAL CELLS IN THE EYE
  6. MIGRATION OF GLIAL CELLS ONTO THE EYE DISC
  7. NEURON–GLIA INTERACTION IN THE EYE DISC
  8. REFERENCES

The intricate interaction between neurons and glial cells is pivotal for the generation and the maintenance of a functional nervous system. During early development, glial cells are often born distant from the place where they eventually settle and neuron–glia interaction provides the most important cues to allow the precise positioning of glial cells. This has been nicely demonstrated in the peripheral nervous system (PNS) of developing zebrafish, where sensory axons navigating to the periphery are followed by Schwann-cell precursors and perineurial cells follow motoraxons as they leave the CNS (Gilmour et al.,2002; Lyons et al.,2005; Kucenas et al.,2008). Quite similar, within the developing embryonic PNS of Drosophila glial cells follow motor neuronal tracts guided by graded adhesiveness along the neuronal tracts (Sepp et al.,2000; von Hilchen et al.,2008; Silies and Klambt,2010). Glial migration has also been studied in other parts of the Drosophila PNS. During wing formation in pupal stages, glial cells originate in the periphery and follow sensory axons toward the CNS (Giangrande,1994; Aigouy et al.,2004, 2008). In all cases, the coordination of neuronal and glial-cell development is of eminent and obvious relevance. In the following, we discuss the current knowledge on how this is regulated in the developing Drosophila compound eye.

Because the first description of the quasi-neurocrystalline lattice made in the developing Drosophila compound eye some almost 40 years ago, the Drosophila eye has been a favorite model system in developmental biology (Wolff and Ready,1993). Many studies provide the molecular framework underlying eye specification, cell-cycle control, cell fate allocation, axon wiring, and pattern formation. Moreover, this organ provided an easy amenable test tube for dissecting many signaling pathways. The knowledge of receptor tyrosine kinase (RTK), Wingless (wg), Hedgehog (Hh), Decapentaplegic (Dpp), and Notch (N) signaling all profited from manipulations in the Drosophila eye (Roignant and Treisman,2009). More recently, even the basis of human neurodegenerative disorders has been successfully studied using the eye model (Bilen and Bonini,2005).

Importantly, and unlike in vertebrates, the eye forms as a peripheral sense organ (Wolff and Ready,1993). All photoreceptor neurons are generated in the eye antennal imaginal disc (from here on simply called eye disc). In contrast, the progenitors of all retinal glial cells are born in the CNS and have to move outward to reach the photoreceptor-cell axons (Choi and Benzer,1994). In the last few years, the compound eye has now also been used to understand glial-cell development. How is the migration of glial cells along their neuronal partners regulated and what controls their subsequent differentiation? In the following, we highlight some molecular mechanisms Drosophila uses to coordinate the development of neurons and glial cells and focus on long-range signaling system.

FORMATION OF THE DROSOPHILA EYE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORMATION OF THE DROSOPHILA EYE
  5. GLIAL CELLS IN THE EYE
  6. MIGRATION OF GLIAL CELLS ONTO THE EYE DISC
  7. NEURON–GLIA INTERACTION IN THE EYE DISC
  8. REFERENCES

The Drosophila eye is a compound eye that develops within the eye imaginal disc. It is specified through an evolutionary highly conserved network of transcription factors centered around the Pax6 encoding genes eyeless and twin of eyeless (Kumar and Moses,2001; Kozmik,2008). Their activity defines the eye anlage and eventually ensures the formation of a compound eye, which harbors about 750 ommatidia [for review, see Silies et al. (2010)]. Each ommatidium comprises eight photoreceptor neurons (R1–R8), which send their axons toward the optic lobe. The growth cones of the R1–R6 axons terminate in the lamina, whereas the growth cones of the R7 and R8 neurons terminate in the medulla (Ting et al.,2007). Thus, glial cells eventually need to support about 6000 axons and neurons in each compound eye (750 ommatidia each with 8 receptor neurons).

The formation of the Drosophila eye occurs in a sequential manner. First, the eye disc is established, which is mounted to the optic lobes via the 12 axons of the larval photoreceptors comprising the Bolwig organ (Meinertzhagen and Hanson,1993). This thin axonal fascicle prefigures the optic stalk and tethers the growing eye disc to the brain until eye eversion in pupal stages. As the vertebrate optic nerve, the optic stalk only contains glial cells and photoreceptor axons. The eye disc is a simple epithelium, which is subsequently structured by the action of only few signaling pathways. The activating ligand of the JAK/STAT pathway is expressed at the posterior most cells and together with Wg and Dpp signaling ensures the correct initiation and progression of neurogenesis in the eye disc (Wang and Huang,2010). Neurogenesis only starts in young third instar eye discs at the very posterior end, close to the point where the fascicle containing the Bolwig axons enters the brain. The first visible sign of the initiation of neuronal development is the formation of the morphogenetic furrow, which sweeps across the eye disc from posterior to anterior during the third instar larval stage (Wolff and Ready,1993). Photoreceptor development begins with the selection of the R8 photoreceptor neurons in the morphogenetic furrow. Every 1.5 h, the morphogenetic furrow releases another row of R8 cells until the entire eye field is structured. Subsequently, R1–R7 and cone cells are added to the developing ommatidia in five sequential steps that are timed almost 5 h apart. Finally, several pigment cells are added during pupal stages (Tomlinson,1988).

The progression of the furrow crucially depends on neuronally expressed Hh and Dpp signaling within the furrow (Roignant and Treisman,2009). For all signaling molecules used to control sequential progression of neurogenesis in the eye disc, it has been shown that they can diffuse in tissue and thus in principle can form gradients (Hooper and Scott,2005; Baker,2007).

GLIAL CELLS IN THE EYE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORMATION OF THE DROSOPHILA EYE
  5. GLIAL CELLS IN THE EYE
  6. MIGRATION OF GLIAL CELLS ONTO THE EYE DISC
  7. NEURON–GLIA INTERACTION IN THE EYE DISC
  8. REFERENCES

Although all photoreceptor neurons are born in the periphery, all glial cells originate from progenitors derived from the CNS or the optic stalk (Choi and Benzer,1994). A series of single-cell labeling experiments by flip out approaches and MARCM studies revealed a set of different glial subtypes in the developing eye anlage, which basically correspond to the set of glial cells as found in other parts of the nervous system (Rangarajan et al.,1999; Hummel et al.,2002; Silies et al.,2007; see Fig. 1). Surrounding the entire nervous system is a thick layer of extracellular matrix called neural lamella. Abutting this ECM are the perineurial glial cells, which define the outer most cell layer of the nervous system (Awasaki et al.,2008; Stork et al.,2008). These cells express the glial-cell marker Repo. Expression of Repo is a frequently used criterion to define glial cells (Campbell et al.,1994; Xiong et al.,1994; Halter et al.,1995). It is only expressed in the nervous system where it is strictly found in non-neuronal cells. The perineurial glial cells, however, are special in the way that they never come in contact with neurons (Stork et al.,2008). Unfortunately, so far, no tool has been developed, which would allow the selective ablation of this cell type. Thus, we can currently only speculate about the function of these cells. This is, in particular, true for the morphologically distinct subtypes of perineurial glial cells at the optic stalk, the eye disc margin, and the leading edge of the migrating perineurial sheath [Fig. 1(D), numbers 1, 2, 4, 5].

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Figure 1. Glial cell types in the eye imaginal disc. The figure shows dissected third instar eye imaginal disc stained for the glial nuclei marker Repo (magenta) and GFP directed by different Gal4 driver strains (green). A: c527-Gal4 driving expression of UAS-laminGFP. Only the perineurial glial cells express the GFP marker. The perineurial glial cells in the optic stalk have a distinct morphology (1) and can be distinguished from the perineurial glial cells in the eye disc (2). B: spinster-Gal4 driving expression of UAS-laminGFP. Only the two subperineurial glial cells express the GFP marker (6). C: Mz97-Gal4 driving expression of UAS-CD8GFP. The morphology of the wrapping glial cells (3) can be seen. D: Schematic summary of the location of the different glial cell types found in the eye imaginal disc. In addition to the cell types shown above are the filopodia bearing perineurial glial cells at the edge of the carpet cell (4) and the glial cells found at the margin of the eye imaginal disc (5).

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Work in the eye imaginal disc has shown that the perineurial glial cells can give rise to wrapping glial cells (Silies et al.,2007). This suggests that these cells define a reserve pool to generate glial cells when required (injury, plasticity, and development). In agreement with this notion is the ability of the perineurial glial cells to divide. It is also interesting to note that except for the eye imaginal disc, where most perineurial glial cells adopt a spindle-shaped morphology, these cells generally form numerous fine filopodia-like processes (Silies et al.,2007; Awasaki et al.,2008; Stork et al.,2008). The function of these glial processes is still unknown. On the eye disc, the filopodia bearing perineurial glial cells are those that contact nascent axons searching for cues triggering the advance of glial migration and cues, which initiate the differentiation into wrapping glial cells.

Below the perineurial glial cells are the subperineurial glial cells. They form highly interdigitated cell contact zones and express prominent septate junction markers. These cells form most of the hemolymph brain barrier (Bainton et al.,2005; Schwabe etal.,2005; Stork et al.,2008; Mayer et al.,2009). Unlike the perineurial glial cells, these cells are not able to divide. However, the nucleus of the subperineurial glial cells is generally huge, which is most likely due to endoreplication. The subperineurial cells cover the entire nervous system and are extremely large and very flat cells which explains why they have not been noticed for so long (Stork et al.,2008). Because of their flat and large size, the subperineurial glial cells of the eye disc had been called carpet cells (Silies et al.,2007). In the developing eye, only two carpet cells cover the entire eye field on the eye disc, which corresponds to about 10,000 epithelia cells. The nuclei of the carpet cells are generally located at the junction of the optic stalk and the eye imaginal disc. They extend their posterior cell margin down to the lamina in the brain. The anterior end of these two gigantic cells grows along the eye disc and closely follows the morphogenetic furrow. The carpet cells are crucial in organizing the correct positioning of the glial cells on the eye disc (see below).

The third main cell type of the eye imaginal disc is the wrapping glia. The wrapping glial cells differentiate similar to the Remak fiber in the mammalian PNS and engulf several ommatidial axons bundles (Nave and Trapp,2008). Interestingly, the ommatidial axon bundles are wrapped as units, and glial cells usually do not contact the central R8 axon but only axon R1–R7 (Franzdottir et al.,2009). Mostly likely wrapping glial cells do not divide. However, we have been able to induce proliferation in the wrapping glia by expressing an activated FGF-receptor specifically in these cells using the nervana2 Gal4 driver strain (Franzdottir et al.,2009). In addition, a few more special glial cell types are found in the optic stalk and the margins of the eye imaginal disc [for review, see Edwards and Meinertzhagen (2010) and Silies et al. (2010)].

MIGRATION OF GLIAL CELLS ONTO THE EYE DISC

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORMATION OF THE DROSOPHILA EYE
  5. GLIAL CELLS IN THE EYE
  6. MIGRATION OF GLIAL CELLS ONTO THE EYE DISC
  7. NEURON–GLIA INTERACTION IN THE EYE DISC
  8. REFERENCES

Choi and Benzer (1994) were the first to describe the migration of the retinal glial cells onto the eye imaginal disc. During first and second instar larval stages, glial cells proliferate and accumulate in the optic stalk. The carpet glial cells have already formed and engulf the Bolwig nerve (Silies et al.,2007). All dividing glial cells are perineurial cells. In first and second instar larval stages, Gilgamesh and Hedgehog signaling is needed to prevent glial cells from entering the developing eye field (Hummel et al.,2002). Gilgamesh encodes a casein kinase that acts in the Wingless signaling cascade (Davidson et al.,2005; Zhang et al.,2006). Thus, the two main signaling pathways that regulate neurogenesis in the eye imaginal discs are implicated in the regulation of glial migration. Only when neurogenesis is initiated in the eye disc, migration of glial cells starts, triggered by Hedgehog expressing photoreceptor neurons (see Fig. 2). A key role in coordinating glial migration with neuronal differentiation is exerted by the carpet cells. As the morphogenetic furrow sweeps across the eye disc, the carpet cells extend their anterior cell margin that always lies just a few cell rows behind the morphogenetic furrow. Between the carpet cells and the thick extracellular matrix are the perineurial cells, which are the migratory glial cells. As soon these cells reach the anterior end of the carpet cell, they come in contact with the nascent photoreceptor neurons. This is first neuron–glia interaction in the eye disc reprograms the perineurial glia and induces its differentiation as wrapping glia. Molecularly, this switch is accompanied by a changing activity of the FGF-receptor Heartless (Franzdottir et al.,2009). This FGF-receptor is broadly expressed by the eye disc glia and initially becomes activated by the FGF8-like ligand Pyramus, which is expressed by the carpet glia [Fig. 2(B), “a”]. The early activation of FGF-receptor provides a mitogenic signal and renders the glial cells in a motile state. Expression of Pyramus does not appear to provide any directional migration cues, because cell clones, which ectopically express Pyramus ahead of the morphogenetic furrow, are not able to attract retinal glial cells over distance. Rather, it appears that Pyramus expressing cells are a permissive substrate for glial migration and that the FGF-8 like protein Pyramus cannot diffuse over long distances (Franzdottir et al.,2009). Thus, the precise role of Pyramus expression ahead of the morphogenetic furrow is not known [Fig. 2(B), “b”]. The first contact of glial cells with axonal processes at the anterior margin of the carpet cell generates a second source of FGF ligand. This be the second FGF8-like ligand in Drosophila, is expressed only in neurons and can also activate the glial expressed Heartless receptor. Given the sequential presentation of FGF8 like molecules, one might expect that low levels of FGF-receptor activation convey a mitogenic signal and trigger glial motility, whereas further activation triggers glial differentiation [Fig. 2, “c”]. This is most likely not the case as ectopic strong activation of Heartless triggers cell division. Moreover, the presentation of Thisbe, which induces glial differentiation, is accompanied by the induction of Sprouty expression. This is induced by direct neuron-glia contact through a still unknown mechanism (Franzdottir et al.,2009). The function of Sprouty is to dampen receptor tyrosine kinase signaling, and thus low levels of FGF-receptor activity appear to favor the development of differentiating wrapping glial cells.

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Figure 2. Neuron-glia signaling in the eye disc. A: Frontal view of the eye imaginal disc. The neuronal cells are shown. B: Orthogonal view. Signaling routes are indicated. (a) The carpet cell expresses FGF8Pyramus and signals to the perineurial glia. (b) FGF8Pyramus is also expressed anterior to the morphogenetic furrow. Its function here is unknown. (c) Photoreceptor neurons express FGF8Thisbe, which instructs glial differntiation. (d) Dpp signaling controls motility of the perineurial glial cells. (e) A still unknown signal regulates the growth of the carpet cell.

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NEURON–GLIA INTERACTION IN THE EYE DISC

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORMATION OF THE DROSOPHILA EYE
  5. GLIAL CELLS IN THE EYE
  6. MIGRATION OF GLIAL CELLS ONTO THE EYE DISC
  7. NEURON–GLIA INTERACTION IN THE EYE DISC
  8. REFERENCES

The formation of neurons in the eye disc and the proliferation and differentiation of glial cells occurs sequentially. Therefore, neurogenesis and gliogenesis need to be closely coupled. Several signaling systems have been identified to act within the eye disc epithelium to control the ordered progression of the morphogenetic furrow. These processes are likely to also affect the development of the glial-cell population. A key cell type involved in this control is the carpet glia. To better understand the molecular mechanisms underlying neuron–glial interaction in the eye imaginal disc, we have therefore screened for genes that are expressed by this gigantic subperineurial glial cell.

One of the genes identified in a classic enhancer trap screen spinster, which appeared as marker of the carpet cell as well as of all other subperineurial glial cells (Yuva-Aydemir et al., 2011). The gene spinster encodes several transmembrane proteins that were previously shown to regulate the maturation of the lysosome in neurons (Nakano et al.,2001; Sweeney and Davis,2002; Dermaut et al.,2005). Loss of Spinster results in defects in glial migration and highlights the integration of several signaling pathways that converge on the different glial cells (Yuva-Aydemir et al., 2011). The function of the Spinster protein is to guide the route of endosomal vesicles via the formation of multivesicular bodies for degradation in lysosomes. This way, Spinster crucially controls the signal intensity. In spinster mutants, late endosomes accumulate and a glial overmigration phenotype develops, where glial cells are found anterior to the morphogenetic furrow. This suggests that signaling pathways that normally control glial migration are overactivated (Yuva-Aydemir et al., 2011).

Gene dose experiments provided further proof for this concept and the reduction of genes required for the transition from early to late endosomes or the transition of late endosomes to lysosomes efficiently suppressed the spinster mutant glial phenotype. Moreover, the Dpp receptor Thickveins accumulates in spinster mutant perineurial cells. Thus, Spinster appears to antagonize Dpp signaling by facilitating the routing of Dpp receptors towards the lysosome (Yuva-Aydemir et al., 2011). This would provide a simple mechanism how progression of the morphogenetic furrow, which is the source of Dpp, could be coupled to the migration of glial cells [Fig. 2(b), “d”].

Endocytosis and vesicle recycling is more generally required in migrating cells. One of the important functions of endocytosis lies in the constant and dynamic remodeling of adhesive contacts. Endocytosis is involved in the re-location of active signaling receptors to the front of the cell in response to extracellular signals (Ulrich and Heisenberg,2009). For example, Integrins from the cell rear can be relocalized to the leading edge, and during border cell migration in the Drosophila ovary, spatial restriction of the receptor tyrosine kinase signaling by endocytosis ensures the localized intracellular response to guidance cues (Jekely et al.,2005; Ezratty et al.,2009). Likewise, glial migration in the embryonic Drosophila PNS is regulated by the fine tuning of Notch signaling via Numb-mediated endocytosis (Edenfeld et al.,2007). Thus, endocytotic trafficking may affect cell migration through several pathways.

In addition to its function in the perineurial glia, spinster is required for the growth of the carpet cells in a cell autonomous manner. Upon ablation of the carpet cells, we noted uncontrolled glial-cell migration onto the eye disc (Silies et al.,2007). Therefore, the block of precocious glial migration in early eye discs is mediated via controlling the growth of the carpet cell. The reduced size of this cell type in spinster mutants may thus account for part of the glial overmigration phenotype. How spinster controls the size of the carpet cells remains to be investigated, and Hedgehog and Wingless signaling pathways comprise attractive candidates (Fig. 2, “e”). The activation of the Dpp receptor Thickveins is not expected to trigger the growth of the carpet cell. Although this cell type strongly expresses Spinster, it does not appear to express the activated Dpp receptor Thickveins nor does it contain activated downstream signaling components.

In conclusion, these studies indicate that the coordination of glial migration and growth of the eye imaginal disc most likely occur at least two levels. On one hand, the eye disc influences the differentiation of the master glial cell of the eye, the carpet cell, by yet to be defined signaling pathways. The carpet glia in turn directly influences the migration of the perineurial glia. On the other hand, the eye disc secretes Dpp at the morphogenetic furrow. This secreted Dpp also acts on the perineurial glial cells, where it promotes their migratory abilities. Importantly, Dpp itself acts as an inducer of motility and does not act as a guidance signal per se.

The function of spinster in controlling glial differentiation is not confined to the carpet cells but is also required in the wrapping glia. Loss of spinster results in a reduced wrapping of axonal membranes (Y. Yuva-Aydemir and D. Engelen, unpublished results). Thus, spinster function appears to be generally needed to extend cellular processes. Weak spin mutants live till adulthood and show an increase in the accumulation of lipofuscin-like materials with age showing similarity to neurodegenerative lysosomal storage diseases (Nakano et al.,2001; Dermaut et al.,2005). Although neurodegenerative diseases are heterogeneous in their pathological symptoms and nature of the underlying genetic aberration, many of them share a common cellular dysfunctions; defects in vesicular trafficking and lysosomal degradation. The involvement of defective vesicular transport in glial cells may cause the neurodegeneration in Niemann-Pick type C (NP-C) disease, which is a lipid storage disorder characterized by progressive neurodegeneration. NPC-1 is a lysosomal protein and is proposed to mediate trafficking of cholesterol and other lysosomal cargo (Ong et al.,2001). Although mutations in Hspin1, the human homolog of spinster, have not been associated with the any human neurodegenerative disorders, the similarities in the molecular and pathological phenotypes suggest that spinster mutants can be used to model for neurodegenerative diseases.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORMATION OF THE DROSOPHILA EYE
  5. GLIAL CELLS IN THE EYE
  6. MIGRATION OF GLIAL CELLS ONTO THE EYE DISC
  7. NEURON–GLIA INTERACTION IN THE EYE DISC
  8. REFERENCES